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Derek Lowe's commentary on drug discovery and the pharma industry. An editorially independent blog from the publishers of Science Translational Medicine. All content is Derek’s own, and he does not in any way speak for his employer.

Aging and Lifespan

Cellular Rejuvenation, For Real

There’s been a good amount of coverage of this paper, which is certainly worth a read. It concerns the “Yamanaka factors”, four proteins whose expression seems to be key for induced pluripotent stem cells and many other cellular programs besides. This latest work shows that cyclic expression of these in adult (indeed, elderly) rodents seems to ameliorate many signs of aging.

The Yamanaka recipe is expression of Oct4, Sox2, Klf4 and c-Myc, so it’s also referred to as OSKM. These are all pretty heavy-hitting proteins, and despite a great deal of work their functions are far from being completely understood. But the connection between aging and stem cell characteristics became clear when the various functions of the OSKM treatment began to be worked out, and many of them matched up with epigenetic processes known to be deficient in aging cells. A number of studies have shown that cells from older animals and those with accelerated-aging phenotypes can apparently be rejuvenated in vitro by the sort of treatment that you would also use to turn adult cells lines back into stem cells.

Some experiments along these lines have also been tried in whole animals, but this seems to be the most comprehensive so far. The authors used a mutant mouse strain with a defective Lmna gene, which animals are known to suffer from premature aging and other disorders (likewise humans with mutations in the homologous gene). They crossed these with another mouse strain that had been engineered with an inducible gene cassette for OSKM expression, producing prematurely aged mice who could have extra OSKM proteins expressed on exposure to doxycycline.

That’s a good way to do it. The tricky part about all these cellular-reprogramming ideas and stem cells in general is that you’re walking just this side of inducing uncontrolled cellular growth, better known as cancer. Early experiments in animals with induced stem cells, in fact, led to notably higher rates of several different tumors, which is one rather large reason why the human stem cell hype of the last ten or fifteen years has been slow to deliver miracle cures. If you just walk into an animal’s genome and put the pedal down on OSKM expression, you are highly likely to get results that you didn’t want. c-Myc, to pick the prime example, has been known for many years as a protein whose overexpression/overactivity is a hallmark of many tumor types, and there have been many attempts (so far without success) to inhibit its actions as a means of cancer therapy. So the idea of actually upregulating it, and the other three, has to be approached with caution.

You also don’t want to blast cells all over the body back into becoming pluripotent stem cells, because who knows what that’s going to lead to. The idea with the doxycycline-driven expression system is to try to reset many of those epigenetic markers, without losing cellular identity or inducing tumor formation, and there’s no real way to know a priori how many times that button has to be pushed (or how many times is too many). A similar inducible OSKM technique had already been shown to lead to cancer in mice, so none of this is idle speculation. And indeed, in this present work if they tried continuously inducing OSKM in these hybrid mice by just putting doxycycline in their drinking water, they only lasted a few days before succumbing to multiple organ failure, probably through just that sort of changing-back-into-stem-cell mechanism just mentioned.

Just inducing expression for shorter periods on a cyclic schedule, though, seemed to work. It’s still a close race, because the mice showed progressive weight loss, which is not normal for rodents and is considered a bad sign. But these animals also showed a significant lifespan increase compared to their untreated cohort – half of them were still alive at an age when all the untreated ones were dead. (Giving doxycycline to the fast-aging mice without the OSKM expression genes did nothing for them, in case you were wondering – a good control to have run). Examination of their various organs and tissues showed unmistakable signs of the treatment, with the OSKM-cycled mice looking far more like wild type than their premature aging phenotype should have allowed. This went all the way from gross pathology, through histopathology examination, and down to specific cellular markers. No signs of tumor formation were seen.

Another marker of aging in tissues and organ systems is a decreased ability to deal with injury and stress. This paper looked also at stressing the pancreas with streptozotocin (STZ), a well-known way to kill off beta-cells and induce insulin insufficiency. Young mice are better able to recover from low-dose STZ exposure than older ones, and OSKM expression, as above, seems to restore the animals’ ability to deal with the loss of beta-cells. Similarly, injury of muscle cells with snake venom cardiotoxin also was tolerated far better in the OSKM-cycled mice, suggesting that repair and regeneration pathways were significantly enhanced.

So when you put all this together, it appears that there’s real promise here that many of the important defects of aging are, in fact, reversible. It’s going to be tricky to realize these effects without stepping over the line into tissue problems and tumorigenesis, as mentioned above, but the important thing about this paper is that it shows that there is a window there that can be targeted. Cycling aged human cells with OSKM expression seemed to lead to similar effects on markers of aging as were seen in the mice cells, so there’s no reason, so far, to think that this is impossible in humans.

Of course, actually doing this in humans will not be easy at all. We’re not quite ready to modify our germ lines to include the OSKM expression cassette in the human genome, nor would we wish to wait several decades while these first modified humans aged to the point that we could see whether putting doxycycline into their drinks would improve them. If we’re going to make people undergo pulsed OSKM expression, we’re going to have to go about it another way, and every possibility comes with its own dangers and unknown factors. It may well be that we’ll have to sneak up on the downstream effects from another direction entirely; it’s too early to say. But at the moment, it looks like this is a goal that’s well worth trying for, and could actually be possible – eventually – to reach.

it’s not clear to me that what’s limiting life-expectancy in WT humans is the same as what limits life-expectancy in these mutant mice. Of course, the experimenters designed a very nice trial in which they could get a signal out. How to get a human therapeutic agent from this (and would that be for progeria? Or would FDA have to define aging as a “disease”?) is far from clear.

Barry, I do like your questions. Interestingly, you were again so close to juvenile hormones. Since, Oct4 has structural resemblance to of hLRH-1 DNA Binding Domain.https://en.wikipedia.org/wiki/Liver_receptor_homolog-1
IMO if you are playing with good toys such OKSM and ask right questions you can be more close to longevity than you are.

Might as well do it. First of all I wouldn’t be too excited about upregulating c-Myc either:

“NO is known to be involved in numerous pathophysiological processes. In
particular, Tyr [tyrosine] nitration of various proteins by peroxynitrite has been detected in a number of inflammatory or degenerative diseases (54, 55), and tumors (9, 56). As such, it has generated enthusiasm for managing diseases with agents that modulate NO signaling pathways.”

“The potential of chemical NO donor sodium nitroprusside (SNP) to induce apoptosis directly from NO liberation has been established in vitro [5]. The fact that NO is capable of triggering apoptosis is consistent with its ability to induce DNA damage, the inhibition of DNA synthesis and cell cycle arrest [6, 7]. The reaction product formed between NO• and superoxide [i.e., peroxynitrite (ONOO-)] plays a critical role in the induction of inflammatory reaction and apoptosis, but is also associated with tumor promotion and/or progression. Potentially toxic levels of peroxynitrite can be achieved whenever NO• and O2.- production is stimulated, due to the fact that a 100-fold increase in the rate of peroxynitrite formation occurs for every 10-fold increase in NO• and O2.- concentration [8].”

It is the overactivation of NMDA receptors that leads to peroxynitrite formation and caspase 3-activation and subsequent neuronal cell death in Alzheimer’s disease and c-Myc may be a part of this process.

Doxycycline should be looked at as an anti-aging drug on its own, as there is a substantial body of literature showing its anti-inflammatory properties and it is the only FDA approved inhibitor of MMPs known to man.
Then there is the fact that the Tet system doesn’t always work well using doxycycline, so there is a derivative that works 10-fold better especially in lipophilic compartments, called 9TB dox, which works way better to express proteins.

And those tetracyclines also inhibit the formation of peroxynitrite, so you better watch out! You better not cry, you better not pout, Im telling you why!

Metabolism and post-translational modifications….it’s the quantum physics of biology that we know even less about. Many studies have shown how metabolism changes with age. Metabolism is directly linked, for example, to the production and regulation of a very interesting PTM known as the ‘O-GlcNAc modification’, which is the addition of a N-acetylglucosamine to SER/THR sites of intracellular proteins–often the same sites as where proteins are phosphorylated. One very well known class of proteins to be modified by O-GlcNAc is the entire set of the core Yamanaka factors that regulate pluripotency.

Metabolism and post-translational modifications….it’s the quantum physics of biology that we know even less about. Many studies have shown how metabolism changes with age. Metabolism is directly linked, for example, to the production and regulation of a very interesting PTM known as the ‘O-GlcNAc modification’, which is the addition of a N-acetylglucosamine to SER/THR sites of intracellular proteins–often the same sites as where proteins are phosphorylated. One very well known class of proteins to be modified by O-GlcNAc is the entire set of the core Yamanaka factors that regulate pluripotency.

O-GlcNAc Regulates Pluripotency and Reprogramming by Directly Acting on Core Components of the Pluripotency Network

The earth is warming at an unprecedented rate – some feel we have already passed the point of no return and entered a positive feedback loop that will accelerate warming even more. The resulting loss of coastal lands from rising ocean levels and increased frequency of severe meteorological disturbances (drought, forest fires, hurricanes, flooding) will cause unprecedented civil unrest, wars, and massive refugee exodus to first world countries, resulting in a severe anti-immigrant backlash and the election of populist demagogues whose unpredictable and misguided policies will cause economic and political chaos and topple the very fragile order of nations. These changes are already well underway.
You want to increase human lifespan ? Work on these problems, rather than haplessly fiddling with genes.

Extending the human lifespan to several centuries is a great way to make people care about problems 200+ years from now.
Also, there’s more than one scientist on Earth. More than one problem can be solved at the same time.

e.g. CRY1/CRY2 KO mice have no circadian rhythm, but still show all of the “cyclic” cell cycle events (cells grow, cells divide). There myriads events inside cells that happen to be cyclic or follow patterns of “what goes up..”, like all PTMs.